Gene-Expression Profiling Of Human Metastatic Non-Small Cell Lung Cancer Cells Enriched By Repeated Intracardiac Injection In Mice

Background: Lung cancer is the worldwide leading cause of death from cancer. Metastasis is a main cause of death in patients with non-small cell lung cancer (NSCLC). The molecular mechanism underlying NSCLC metastases is largely unknown. Although genome-wide comparison of gene expression between patients9 primary and metastatic tumors may aid to identify metastasis-associated genes and be of more clinical relevance, the primary/metastatic tumor pairs are often difficult to obtain or not available. Therefore we performed a genome-wide study using highly metastatic NSCLC cell clones derived by repeated intracardiac injection in mice. Methods: At first for the enrichment of brain metastatic activity of NSCLC cells, we performed 3 rounds of intracardiac injection of A549 lung adenocarcinoma cell line into Athymic Ncr Nu/Nu mice. Brain metastases were confirmed by monitoring the outgrowth of A549 cells in cultured brain tissues. At round 1, 6 out of 22 mice developed brain metastases, whereas at rounds 2 and 3, 16/22 and 10/10 mice developed brain metastases respectively, demonstrating a progressive increase of brain metastases of A549 cells by repeated injections. To determine if the 100% metastatic penetrance at round 3 is brain tropism or a simple reflection of increased invasion/colonization capacity of A549 cells throughout the body, we generated A549-round 3 cells expressing luciferase. On the subsequent non-invasive bioluminescence imaging of luciferase-expressing A549-round 3 cells after intracardiac injection , 4 out of 4 mice developed multiple metastases including to brain and bones, suggesting that repeated intracardiac injection/brain metastatic selection augments metastases not only to the brain but also to other tissues or organs. Gene expression profiling of triplicate samples of A549 parental, round 1,2, and 3 cells, was performed using Affymetrix GeneChip Human Gene 1.0 ST arrays. Public gene expression data of PC9 lung adenocarcinoma cell line parental and brain metastatic round 3 derived from repeated intracardiac injection were obtained from GEO (GSE14107). Using these two microarray data sets, differentially expressed genes in metastatic round 3 were analyzed and confirmed with real time RT-PCR. Results: With the cut-off of FDR less than 0.1 and fold change larger than 1.5, 48 differential genes were identified. Further literature-based search narrowed down the number of genes to 11 cancer-associated genes: LAMC2, AXL, MYBL1, CYR61, CTGF, TGM2, and FSCN1 were up-regulated whereas ASPM, CCNG2, PDCD4, and CD24 were down-regulated in round-3 metastatic cells. Real time RT-PCR confirmed 5 up-regulated genes (LAMC2, AXL, MYBL1, CYR61, and CTGF) as metastasis-promoting genes. Conclusions: Our gene-expression profiling using homogeneous highly metastatic cell clones through in vivo enrichment identified five metastasis-promoting genes. Functional characterization of those genes in NSCLC metastasis is on the way and will be discussed. Note: This abstract was not presented at the conference. Citation Format: Yongwha Moon, Sami Sarfaraz, Donna Voeller, Lynn Young, Trung Pham, Kang-Seo Park, Yisong Wang, Giuseppe Giaccone. Gene-expression profiling of human metastatic non-small cell lung cancer cells enriched by repeated intracardiac injection in mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B96.